Borrelia burgdorferi is the causative agent of Lyme disease in North America and is transmitted by ticks of the genus Ixodes. It is a highly invasive spirochete that can cause infection and manifestations in humans and other mammals that persist for months to years. The infection has localized, disseminated, and persistent phases, and B. burgdorferi appears to cause dermal, neurologic, cardiovascular, and arthritic symptoms primarily though the ability to invade almost any tissue, establish long-term infection, and induce inflammatory responses. The bacterium produces no known toxins, and its mechanisms of pathogenesis are largely unknown. Genetic studies using low-passage, infectious B. burgdorferi have been challenging due to exceedingly low transformation rates and plasmid loss; as a result, fewer than 50 genes have been investigated by allelic exchange or other site-directed mutagenesis methods for their importance in the mammal-tick infectious cycle. During the prior grant period, a sequence-defined library of 4,479 signature-tagged mutagenesis (STM) transposon mutants was generated in a transformable, infectious clone of B. burgdorferi B31. The plasmid content of the clones in this library was also determined using a novel Luminex-based high throughput strategy. Using the library, we have already performed STM screening of mouse infectivity of 484 transposon mutant clones in 434 different genes. In Aim 1, we will complete the systematic analysis of the roles of the 790 mutated protein-encoding genes in the infection of C3H/HeN mice using the STM mutant library. For this analysis, we will employ the STM Luminex Assay protocol developed during the prior grant period to analyze each mutant clone in 3 mice per group, 5 different tissues, and two time points in a high throughput manner. The goal of Aim 2 is to determine the effects of the transposon mutations on infectivity, persistence and transmission of B. burgdorferi in Ixodes scapularis ticks. Groups of the STM mutants will be transmitted to mice by larval ticks infected by immersion with a mixture of organisms with different mutations and signature tags. The survival of the STM clones will be analyzed before and after feeding on mice, and transmission of the clones to the mice will also be determined by the STM Luminex Assay. In Aim 3, the findings in Aims 1 and 2 will be used to select classes of mutants for detailed infectivity, complementation, and functional analysis. These analyses will include studies of genes involved in chemotaxis, motility, nutrient transport, surface proteins involved in adherence and other roles, novel gene regulation pathways, and additional gene sets identified during the STM screening procedure. The STM library will also be made available to any interested investigator through BEI-Resources, providing maximal resource sharing and stimulation of Borrelia research. We anticipate that this project will continue to fuel new discoveries useful in improving the diagnosis, treatment, and prevention of Lyme borreliosis.